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Mantidis Oötheca (mantis egg case) original species identification via morphological analysis and DNA barcoding
Journal Pre-proof Mantidis Oötheca (mantis egg case) original species identification via morphological analysis and DNA barcoding Jun-Ho Song, Ji-Min Cha, Byeong Cheol Moon, Wook Jin Kim, Sungyu Yang, Goya Choi PII:
S0378-8741(19)32336-0
DOI:
https://doi.org/10.1016/j.jep.2020.112574
Reference:
JEP 112574
To appear in:
Journal of Ethnopharmacology
Received Date: 12 June 2019 Revised Date:
3 December 2019
Accepted Date: 13 January 2020
Please cite this article as: Song, J.-H., Cha, J.-M., Moon, B.C., Kim, W.J., Yang, S., Choi, G., Mantidis Oötheca (mantis egg case) original species identification via morphological analysis and DNA barcoding, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/j.jep.2020.112574. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Mantidis Oötheca species identificaiton
(Original research articles)
Mantidis Oötheca (mantis egg case) original species identification via morphological analysis and DNA barcoding
Jun-Ho Song, Ji-Min Cha, Byeong Cheol Moon, Wook Jin Kim, Sungyu Yang, Goya Choi*
Herbal Medicine Reseources Research Center, Korea Institute of Oriental Medicine, Naju, 58245, Republic of Korea
*Corresponding author: Telephone: +82-61-338-7118 Fax: +82-61-338-7135
E-mail addresses: [email protected] (J.-H. Song), [email protected] (J.-M. Cha), [email protected] (B. C. Moon), [email protected] (W. J. Kim), [email protected] (S. Yang), [email protected] (G. Choi).
Running title: Mantidis Oötheca species identification
Number of pages: 24 Number of figures: 6 Number of tables: 4 1
Mantidis Oötheca species identificaiton
Abstract Ethnopharmacological relevance: Mantidis Oötheca (mantis egg case; sangpiaoxiao) is a medicine from an insect source, which has been widely used in Asian countries. However, misidentification due to a lack of information given variations in the medicinal portion of the ootheca and morphological similarities of the ootheca as an egg chamber. Aim of the study: Thus, this study aims to provide the first comprehensive data for discriminating authentic of Mantidis Oötheca. Here, we provide detailed ootheca morphology and their molecular information to accurately identify Mantidis Oötheca. Materials and methods: Oothecae of Tenodera angustipennis (Saussure, 1869), Tenodera sinensis (Saussure, 1871), Hierodula patellifera Serville, 1839, and Hierodula sp. were used in the comparative morphological, principal component analysis, and DNA barcoding. Results: The morphological analyses revealed that the emergence area, outline, angle of distal end, width of air-filled layer, and weight are useful diagnostic characters. Using these quantitative and qualitative characteristics, we developed the effective identification key. Furthermore, our CO1 sequences from all individuals were monophyletic with high bootstrap values at genus and species levels. Moreover, morphological identification using our developed key among all studied individuals agreed with molecular identification results using CO1 barcoding data. Conclusions: These multilateral approaches, including morphological, statistical, and DNA barcoding methods are highly reliable identification tools. Moreover, our diagnostic key characteristics and molecular barcoding should aid in the accurate identification, authentication, and quality control of Mantidis Oötheca medicinal materials.
Keywords: Mantidis Oötheca, Tenodera, Hierodula, PCA, CO1 barcoding, identification. 2
Mantidis Oötheca species identificaiton
1. Introduction As a group, insects comprise about 70% of all living organisms (Gullan and Cranston, 2005). Insects and insect-derived products have been used as sources of food, medicine, and chemical materials for a long time on account of their great diversity and richness. In particular, medicinal insects and their derived products are used to treat various diseases, a practice called entomotherapy (Costa-Neto, 2005; Fan and Ding, 2001; Feng et al., 2009). Insect bodies, eggs, egg shells, exuviae, and secretions are still used in traditional medicine (Feng et al., 2009). Traditional medical practices in Asian countries, including Korea, China, and Taiwan, commonly use mantis ootheca (i.e., an egg shell, egg chamber, or egg case), which is a complex structure that female praying mantises provide to support and protect their eggs from adverse environmental conditions and natural predators (Kramer, 1973). According to the Korean Herbal Pharmacopoeia, authentic Mantidis Oötheca refers to only the steamed ootheca of Tenodera angustipennis (Saussure, 1869) (tribe Polyspilotini), Statilia maculata (Thunberg, 1784) (tribe Mantini), or Hierodula patellifera Serville, 1839 (tribe Paramantini), all of which belongs to the Mantidae family (Korea Food and Drug Administration, 2014; Korea Institute of Oriental Medicine, 2019). The Pharmacopoeia of the People’s Republic of China and the Taiwan Herbal Pharmacopoeia have designated the ootheca of Tenodera sinensis (Saussure, 1871) (tribe Polyspilotini), Statilia maculata (Thunberg, 1784), and Hierodula patellifera Serville, 1839 as authentic Mantidis Oötheca (Sangpiaoxiao), called "Tuanpiaoxiao", "Changpiaoxiao", or "Heipiaoxiao", respectively (Chinese Pharmacopoeia Commission, 2015; Committee on Chinese Medicine and Pharmacy, 2013; Korea Institute of Oriental Medicine, 2019). The differences between the authentic species, which are designated in the Pharmacopoeias of each country, result in the 3
Mantidis Oötheca species identificaiton
distribution of adulterants or counterfeits in markets (National Institute of Food and Drug Safety Evaluation, 2012). Traditionally, Mantidis Oötheca have been used for the clinical treatment of incontinence and frequent urination (Korea Institute of Oriental Medicine, 2019). Modern pharmacological studies have shown that this medicinal insect has anti-diuretic (Jia and Jia, 2016; Tan et al., 1997), anti-oxidant, anti-atherosclerotic (Ming-zhe, 2012), and vasorelaxation (Kim et al., 2016) properties. However, a number of reports have emerged regarding the medicinal portion of the ootheca that indicate misidentifications due to the great variety of mantis ootheca available that are morphologically similar (National Institute of Food and Drug Safety Evaluation, 2012; Wen et al., 2013). Moreover, mixed oothecae from different species within one package are currently sold in commercial markets. Thus, it is necessary to provide accurate information regarding the species present in Mantidis Oötheca in order to ensure quality. Previous studies on the relationship between Mantis, Mantidis Oötheca (Wen et al., 2013), and identification based on DNA barcoding (Wang et al., 2015) have been conducted. However, oothecae morphology, which contains great potential for taxonomic and systematic classification, has not yet been studied. Therefore, we carried out this study to (1) provide detailed descriptions and illustrations of the morphology of Mantidis Oötheca, including adulterants, to create a morphological identification key, (2) to determine phylogenetic relationships using universal DNA barcode sequences, and (3) to evaluate the relationship between morphology and molecular data for accurate identification.
2. Materials and Methods 4
Mantidis Oötheca species identificaiton
2.1. Medicinal Materials Oothecae of Tenodera angustipennis and T. sinensis (Tuanpiaoxiao), which have been designated as authentic herbal materials in the Korean Herbal Pharmacopoeia (KHP) and the Pharmacopoeia of the People’s Republic of China (ChP), respectively, were used in the analysis. The oothecae of Hierodula patellifera (Heipiaoxiao), which is described as Mantidis Oötheca both the KHP and ChP, and Hierodula sp., which is currently mixed with adulterant species in medicinal markets due to morphological similarities, were also included. Ootheca terminology and identification follows Breland and Dobson (1947), Chinese Medicinal Material Images Database (2012), and Brannoch et al. (2017). All medicinal materials were purchased in medicinal markets from commercial suppliers and samples were deposited in the Korean Herbarium of Standard Herbal Resources (Index Herbariorum code KIOM) at the Korea Institute of Oriental Medicine, Naju, Korea (T. angustipennis, 2-19-0001, 2-19-0002; T. sinensis, 2-09-0025; H. patellifera, 2-19-0003A; and Hierodula sp., 2-19-0003B.
2.2. Morphological Analysis Measurements and optical observations of 20 randomly selected oothecae from each of the five vouchers were recorded for a total of 100 oothecae. A set of digital Vernier calipers (CD-15CP, Mitutoyo, Kawasaki, Japan) was used to measure the length (LG), width (WD), thickness (TH), length of emergence area (LE), width of emergence area (WE), and width of air-filled layer (AC; Fig. 1). Furthermore, a digital protractor was used to measure angle of distal end (DA). The general shape, cross-sectional shape, color, the number of egg chambers (NE), emergence area, and the flaps were recorded using an optical and a stereomicroscope (Olympus SZX16, Olympus, Tokyo, Japan). Ootheca cross-sections were prepared by cutting down the middle of the ootheca using a single-edge blade (DN-52, 5
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Dorco, Seoul, Korea), and images were captured using a digital camera. The weight of the ootheca (WG) was measured using an electronic precision balance (PS 1000.R1, RADWAG Balances & Scales, Radom, Poland).
2.3. Principal Component Analysis A principal component analysis (PCA) was performed to verify whether the quantitative morphological data allowed species grouping. The first three principal components (PCs) with eigenvalues larger than one were represented. The results were presented in a two-dimensional plot of the first and second principal components. These analyses were performed using the program PC-ORD version 5.31 (McCune and Mefford, 2011). The PCA analysis was performed with eleven variables (LG, WD, TH, LE, WE, LE/LG, WE/WD, NE, DA, AC, and WG).
2.4. Preparation of Genomic DNA All medicinal materials were identified based on morphological characteristics and total genomic DNA was extracted from 60 individuals (Appendix). DNA was extracted using a DNeasy® Blood and Tissue Kit (Qiagen, Valencia, CA, USA) according to the protocol. The purity and concentration of extracted DNA were assessed using a spectrophotometer (Nanodrop ND-1000, Nanodrop, Wilmington, DE, USA) and 1.5% agarose gel electrophoresis. The final DNA concentration used for PCR amplification was approximately 15 ng/µL in TE buffer. Extracted DNA samples were stored at -20°C (Kim et al., 2016).
2.5. PCR Amplification of CO1
6
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Using the primers (CO1-C02 5′-AYT CAA CAA ATC ATA AAG ATA TTG G-3′ and CO1-C04 5′-ACY TCR GGR TGA CCA AAA AAT CA-3′) developed by Che et al. (2012) a fragment of the mitochondrial cytochrome oxidase subunit 1 (CO1) was amplified. PCR amplifications were performed in 40 µL reaction volumes containing 10 mmol/L TrisHCl (pH 9.0), 2.5 mmol/L MgCl2, 200 µmol/L each dNTP, 10 mmol/L (NH4)2SO4, 0.5 U Taq DNA polymerase (Solgent, Daejeon, Korea), 0.5 µmol/L each primer, and approximately 15 ng of template DNA (Kim et al., 2016). PCR amplification was performed using a DNA Engine Dyad® PTC-0220 (Bio-Rad, Foster City, CA, USA). The modified parameters were as follows: 95°C for 5 min; 35 cycles of 1 min at 95°C, 1 min at 45°C, and 1 min at 72°C; and a final extension for 5 min at 72°C (Kim et al., 2016). PCR products were separated using 1.5% agarose gel electrophoresis with a 100-bp DNA ladder (Solgent, Daejeon, Korea).
2.6. Nucleotide Sequence and Phylogenetic Analysis Amplified mitochondrial CO1 DNA fragments were rescued from agarose gels using a QIAquick® Gel Extraction Kit (Qiagen, Valencia, CA, USA) and subcloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). Inserted fragments were sequenced in both directions using an automatic DNA sequence analyzer (ABI 3730, Applied Biosystems Inc., Foster City, CA, USA; Kim et al., 2016). The samples of Hierodula patellifera (11 individuals; abbreviation HP), Hierodula sp. (12 individuals; HS), Tenodera angustipennis (29 individuals; TA), and T. sinensis (8 individuals; TS) were used in the analysis (Appendix). To construct the phylogenetic tree, CO1 sequences of eight related species were downloaded from NCBI GenBank: H. formosana (Giglio-Tos, 1912), KR703238; H. patellifera, KX611803; Hierodula sp., KR011164; Mantis religiosa (Linnaeus, 1758), FJ802846; Statilia 7
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apicalis (Saussure, 1871), KR011166; S. maculata, KT036560; T. angustipennis, KR052038; T. sinensis, KY689132. The 694-bp CO1 sequences were assembled and edited using BioEdit version 7.2.5 (Hall, 1999). The contigs were aligned to analyze intra- and inter-species variations in the sequences. For distance analyses, pairwise sequence divergences were calculated using the Kimura-2-parameter (K2P) distance model in MEGA 6 software. The phylogenetic analysis based on the entire CO1 sequences was performed by MEGA 6 version 6.06 (Kimura, 1980; Tamura et al., 2013). The phylogenetic tree was constructed using the NJ method with the K2P model as recommended by Hebert et al. (2003a), the pairwise deletion for gaps/missing data treatment, and 1000 replications for bootstrapping with Acrida cinerea (Thunberg, 1815) (NC014887) as an outgroup.
3. Results 3.1. Morphological Characteristics The morphological variation of the ootheca of four species is described. The representative and quantitative ootheca characteristics of all species evaluated are summarized in Table 1. Representative oothecae of the four species are illustrated in Figs. 1– 3. The oothecae of studied species were fusiform (Fig. 2A), barrel-like (Fig. 2D), and ellipsoid (Fig. 2G, J) in the dorsal view, and obovate (Fig. 2C) and mostly circular to orbicular (Fig. 2F, I, L) the cross- sectional view. The proximal end of all studied ootheca was either partially or fully encircling the substrate to which it was attached (usually a branch, branchlet, or stem) from the proximal to nearly the distal end in such a way that the ootheca laid with its ventral surface hidden, and the emergence area was almost parallel to the 8
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substrate. Tenodera sinensis possessed the largest ootheca (27.9 × 23.7 mm average area; Fig, 2D–E), whereas T. angustipennis had the smallest ootheca among species studied (33.84 × 9.84 mm; Fig. 2A–B). The external wall color ranged from light brown to yellowish brown (Fig. 2D–E) or reddish dark brown to dark brown (Fig. 2A–B, G–H, J–K). In addition, the external wall was either lustrous (Fig. 2G–L) or lusterless (Fig. 2A–F), soft or hard in strength, and smooth or sponge-like in texture. The studied oothecae contained 13–33 egg chambers that were aligned one after another in zigzag arrangement (Fig. 3A, C, E, G). The emergence area exhibited a single emergence opening (Fig. 3G) or a flexible flap (Fig. 3A, C, E).
3.2. Description of Four Species Ootheca Tenodera angustipennis (Sangpiaoxiao). Ootheca fusiform (i.e., proximal end obtuse, distal end tapered; Fig. 2A, B) and mostly obovate in cross-section (Fig. 2C). Ventral part of ootheca formed a furrow from the proximal to distal end (Fig. 2C). External wall dark brown in color, somewhat lusterless and somewhat hard in strength. External coating is a brownish textured. Exhibiting 19–33 egg chambers whose boundaries are clearly delimited, visible on the lateral view as parallel ridges that obliquely arrange on the ventral furrow (Fig. 3B). Distal end of ootheca tapered, dorsoventrally compressed and slightly inclined in the lateral view (Fig. 2B). Emergence area composed of multiple openings, significantly elevated, and all aligned to form a zigzag arranged in two parallel rows and along the dorsal longitudinal axis of the ootheca. Each egg chamber exhibits a flexible flap (i.e., an operculum) that partially closes its corresponding opening. These flaps project slightly beyond the edge of the ootheca; the apex of flaps is rounded (Fig. 3A). Measurements (Table 1): length, 23.81–40.44 mm; width, 7.80–11.54 mm; thickness, 7.50–11.96 mm; length of emergence area, 18.10–30.30 mm; width of emergence area, 3.07– 9
Mantidis Oötheca species identificaiton
5.47 mm; number of egg chambers, 19–33; angle of distal end, 22.1–50.1°; width of air-filled layer, 0.29–1.03 mm; weight, 0.16–0.62 g.
Tenodera sinensis (Tuanpiaoxiao). Ootheca barrel-like (Fig. 2D, E) and mostly circular to orbicular in cross-section (Fig. 2F). Ventral part of ootheca formed a wide furrow from the proximal to distal end (Fig. 2F). External wall light brown to yellowish brown in color, somewhat lustrous, soft in strength, and sponge-like in texture. Exhibiting 16–26 egg chambers whose boundaries are indistinct, visible on the lateral view as parallel ridges that almost perpendicularly arrange to the ventral furrow (Fig. 3D). Distal end of ootheca truncated, rough, and steep in incline in the lateral view (Fig. 2E). Emergence area composed of multiple openings, all aligned to form a nearly zigzag pattern, arranged in two parallel rows along the dorsal longitudinal axis of the ootheca. Each egg chamber exhibits a flexible flap that completely closes its corresponding opening. These flaps project slightly beyond the edge of the ootheca, and the apex of flaps is rounded or obtuse (Fig. 3C). Measurements (Table 1): length, 19.62–34.03 mm; width, 20.19–27.37 mm; thickness, 13.14–25.26 mm; length of emergence area, 16.20–31.40 mm; width of emergence area, 4.60–8.98 mm; number of egg chambers, 16–26; angle of distal end, 63.6–98.7°; width of air-filled layer, 3.69–8.59 mm; weight, 0.49–1.14 g.
Hierodula patellifera (Heipiaoxiao). Ootheca ellipsoid (Fig. 2G) and mostly circular in cross-section (Fig. 2I). Ventral part of ootheca formed furrow from the proximal to distal end (Fig. 2I). External wall reddish dark brown to reddish brown in color, lustrous, hard in strength, and smooth. Exhibiting 14–21 egg chambers whose boundaries are clearly delimited, visible in the lateral view as parallel ridges that are obliquely arranged on the ventral furrow (Fig. 3F). Distal end of ootheca slightly tapered, with a moderate incline in the 10
Mantidis Oötheca species identificaiton
lateral view. Emergence area composed of multiple openings, all aligned to form a zigzag arranged in two parallel rows along the dorsal longitudinal axis of the ootheca. Each egg chamber exhibits a flexible flap that completely closes its corresponding opening. These flaps project beyond the edge of the ootheca, the apex of flaps is obtuse (Fig. 3E). The flaps of the distal end of ootheca are situated upwards (Fig. 2H). Measurements (Table 1): length, 20.23–30.26 mm; width, 10.23–14.04 mm; thickness, 9.26–12.5 mm; length of emergence area, 12.05–23.67 mm; width of emergence area, 3.40–5.42 mm; number of egg chambers, 14–21; angle of distal end, 52.8–69.6°; width of air-filled layer, 0.25–1.63 mm; weight, 0.41–0.73 g.
Hierodula sp. Ootheca ellipsoid (Fig. 2J) and mostly circular in cross-section (Fig. 2L). Ventral part of ootheca formed furrow from the proximal to almost the distal end (Fig. 2L). External wall reddish dark brown to dark brown in color, lustrous, very hard in strength, and smooth. Exhibiting 13–21 egg chambers whose boundaries are clearly delimited, visible in the lateral view as parallel ridges that are obliquely arranged on the ventral furrow (Fig. 3H). Distal end of ootheca truncated, rough, with a steep incline in the lateral view (Fig. 2K). Emergence area slightly elevated and composed of approximately 25–42 openings, all aligned to form a zigzag arranged in two parallel rows along the dorsal longitudinal axis of the ootheca (Fig. 3G). Measurements (Table 1): length, 20.95–29.93 mm; width, 11.60–14.09 mm; thickness, 12.57–15.14 mm; length of emergence area, 17.95–27.40 mm; width of emergence area, 3.56–4.94 mm; number of egg chambers, 13–21; angle of distal end, 79.3–99.2°; width of air-filled layer, 0.42–0.94 mm; weight, 0.81–1.35 g.
3.3. Principal Component Analysis (PCA) 11
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The relationships among the species evaluated with quantitative ootheca morphological data were explored using a PCA analysis (Fig. 4). The first two PCs explained 70.16 % of the total variance of the analyzed data (Table 2). The first principal component (PC 1) accounted for 49.88 % of the variance based on the values of the ootheca size (LG, WD, TH, DA), emergence area size (WE, WE/WD), ootheca shape (DA), and weight (WG; Table 2). The second principal component (PC 2) explained 20.27 % of the data variability, of which length of emergence area (LE) and the number of egg chambers (NE) were the significant variables for the ordination of species (Table 2). The PCA biplot reveals the groupings of the operational taxonomic units (OTUs) that correspond to each species. For the species T. angustipennis, the same species OTUs but different vouchers, are grouped on the positive side of the PC 1 axis, while all OTUs of T. sinensis are grouped on the negative side of the PC 1 axis (Fig. 4). Most of the OTUs of both H. patellifera and Hierodula sp. are positioned on the positive side of the PC 2 axis. However, OTUs of H. patellifera are positioned on the central to positive side of the PC 1 axis, while and Hierodula sp. OTUs are positioned on the positive side of the PC 1 axis with regard to the data from DA (Fig. 4).
3.4. Identification Key Based on Ootheca Morphology 1. Ootheca barrel-like in shape; external wall light brown to yellowish brown in color, with sponge-like texture; width of air-filled layer, 3.69–8.59 mm ------------------------------------------------------------------------------------------------------------ Tenodera sinensis (Saussure, 1871) – Ootheca fusiform or ellipsoid in shape; external wall reddish dark brown to reddish brown in color, and not sponge-like in texture; width of air-filled layer, 0.25–1.63 mm -------------- 2 2. Ootheca fusiform in shape (distal end tapered); external wall lusterless, brownish textured; angle of distal end 22.1–50.1° -------- Tenodera angustipennis (Saussure, 1869)
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– Ootheca ellipsoid in shape (distal end not tapered); external wall lustrous, smooth; angle of distal end 52.8–99.2° --------------------------------------------------------------------------- 3 3. External wall hard in strength; each egg chamber exhibits a flexible flap; angle of distal end 52.8–69.6°; weight 0.41–0.73 g ---------- Hierodula patellifera Serville, 1839 – External wall very hard in strength; each egg chamber exhibits a single emergence opening; angle of distal end 79.3–99.2°; weight 0.81–1.35 g ---------------- Hierodula sp.
3.5. Genetic Variability and Phylogenetic Relationships The length of the CO1 region was 694-bp in over 60 of the ootheca studied (Table 3, Fig 5, see Appendix for accession numbers). The intra-specific genetic sequence variability was 0.10% for Hierodula sp., 0.16% for Tenodera angustipennis, 0.20% for H. patellifera, and 0.41% for T. sinensis (Table 3). Inter-specific genetic distance values ranged from 0.50 (Statilia maculata-S. apicalis) to 18.12% (T. angustipennis-S. maculata; Table 4). The interspecific genetic distances of each congeneric species were 0.50% in Statilia and 7.22% in Tenodera. Hierodula sp. is genetically closer to H. formosana (1.93%) than H. patellifera (13.67%) (Table 4). The phylogenetic tree constructed using the neighbor-joining (NJ) method revealed that all samples (operational taxonomic units) were clustered monophyletically with high bootstrap values at the species level (100%; Fig. 6). Within the studied species, this clade is divided into two distinct and well-supported; one of the clades contained T. angustipennis and T. sinensis, while the other contained H. patellifera, H. formosana, and Hierodula sp. (Fig. 6). Within the genus Hierodula, H. formosana were closely related to Hierodula sp. (levels of a genetic distance value, 1.93%; Table 4, Fig. 6).
4. Discussion
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The ootheca characteristics of the species evaluated show diversity in general shape, texture, strength, the width of the air-filled layer of the external wall, and the type of emergence opening present (Table 1). Although mantis oothecae have been used for important medicinal purposes as Mantidis Oötheca, their macroscopic structure remains largely understudied. Moreover, misidentification and incorrect information exists with regard to this product (National Institute of Food and Drug Safety Evaluation, 2012). Misuse of traditional medicines due to adulterants or counterfeits may even result in severe harm to patients (Zhao et al., 2007). Thus, the accurate identification of authentic Mantidis Oötheca is necessary to safeguard their clinical effects. Morphological characteristics of ootheca were first used in taxonomic studies (Breland and Dobson, 1947). Since the time of Breland and Dobson (1947), ootheca characteristics have been currently used for classifying taxa (Brannoch et al., 2017; Rivera and Svenson, 2016). In particular, Brannoch et al. (2017) suggest that the following relevant diagnostic features should be included in species descriptions: shape, size, external wall, point of attachment, egg chambers, and emergence area. The morphological variation of ootheca from this study agrees with those of previously published data from specimens collected from America (Breland and Dobson, 1947). The emergence area of T. angustipennis, with a significantly elevated ridge (Fig. 2B–C), resembles that of the American oothecae of the same species (Breland and Dobson, 1947, Plate V, Fig. 2). Moreover, oothecae of T. sinensis from America (Breland and Dobson, 1947, Plate VI, Fig. 1), which are characterized by larger ootheca mass, globular outline (i.e., barrel-like), and evident air spaces (i.e., width of air-filled layer), were also similar to those of the present study (Fig. 2D–F). Thus, these retained morphological characteristics, which show no geographical variation, could represent important and stable diagnostic keys. Oothecae of Hierodula sp., which are morphologically similar to those of an authentic species H. patellifera, showed single 14
Mantidis Oötheca species identificaiton
emergence opening (Fig. 3G) with a distal angle of approximately 80–100° (Fig. 2K) and were more than 0.8 g per sample heavier than those of H. patellifera. These data suggest that the presence of flexible flap, the angle of distal end, and weight per ootheca are convenient and useful characteristics that can be used for the immediate identification of medicinal insect ootheca. Moreover, our PCA analysis based on the data matrix revealed that the OTUs of each species were well-clustered (Fig. 4). This data matrix, which includes the useful quantitative data of correctly identified species, gives us objective evidences to identify the oothecae. Thus, our data matrix and statistic techniques might be effective tools for more accurate identification of medicinal ootheca. Oothecae of S. maculata, known as a "Changpiaoxiao", has been listed in the Pharmacopoeia of Asian countries including Korea, China, and Taiwan (Korea Institute of Oriental Medicine, 2019). However, we found that the ootheca of S. maculata was not distributed in Korean pharmaceutical and herbal medicinal markets through our market survey. Moreover, morphological characteristics such as shape and color of the oothecae, differ from our studied oothecae (Patel, 2018). The mitochondrial genome is a powerful molecular marker (Boore, 2006; Hirase et al., 2016). In particular, the cytochrome c oxidase 1 (CO1) sequence was sufficient to generate DNA barcodes for species identification (Hebert et al., 2003a, b, 2004a, b). In order to evaluate the origin and evolution of Mantodea, phylogenetic studies using mitochondrial genomes have recently been conducted (Ye et al., 2016; Zhang and Ye, 2017; Zhang et al., 2018). In addition, molecular identification using the CO1 sequence has also been evaluated (Wang et al., 2015). Our sequence data from all individuals of each species were monophyletic with high bootstrap values at the genus and species levels (Fig. 6). This kind of phylogenetic tree agrees 15
Mantidis Oötheca species identificaiton
with previous results (Wang et al., 2015). Moreover, the morphological identification among all individuals studied corresponds perfectly with the molecular identification results using COI barcoding data. Interestingly, we found that two morphologically different oothecae are mixed together and sold in medicinal markets. Based on the presence of a flexible flap, the angle of distal end, and weight per ootheca, one was identified as a H. patellifera and the other was identified as Hierodula sp. According to the Wang et al. (2015) phylogeny, clade 3 (H1~H5), which is monophyletic, verifies Hierodula sp. not H. patellifera; although they did not recognize morphological differences with the oothecae of H. patellifera, oothecae were divided into two branches based on CO1 (Wang et al., 2015). Our sequence data of Hierodula sp., which have single emergence opening egg chamber, a 79.3–99.2° angle of the distal end, and are 0.81–1.35 g, are identical with Hierodula sp. from Wang et al. (2015). Moreover, we found that Hierodula sp. is more closely related to H. formosana than H. patellifera. Thus, using these morphological and molecular data, further study is required to accurate identify the species of origin of Hierodula sp., which is currently adulterated with H. patellifera.
5. Conclusions We fully described the morphology of the four ootheca comprising Mantidis Oötheca. The morphological and statistical analysis revealed that the emergence area, outline, angle of distal end, width of air-filled layer, and weight are useful diagnostic characteristics. Moreover, most morphological characteristics are consistent with the species level and correspond with CO1 barcoding data. The diversity of morphological characteristics may be considered as an identification marker for distinguishing authentic Mantidis Oötheca. Our morphological and molecular data should be help in the accurate identification and quality control of Mantidis Oötheca. In addition, these data will be valuable for future research to understand the
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evolution of these characteristics in a phylogenetic context and the ecology of the praying mantises.
Conflicts of interest The authors declare that they have no conflict of interest or competing financial interests related to the publication of this paper.
Acknowledgment This work was supported by the Establishment of Application Base for Chung-bu Medicinal Materials Described in the Dong Ui Bo Gam from the Korea Institute of Oriental Medicine [grant number KSN1812410].
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Mantidis Oötheca species identificaiton
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Feng, Y., Zhao, M., He, Z., Chen, Z., Sun, L., 2009. Research and utilization of medicinal insects in China. Entomol. Res. 39 (5), 313–316. https://doi.org/10.1111/j.17485967.2009.00236.x. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3 (5), 294–299. Gullan, P.J., Cranston, P.S., 2005. The Insects an Outline of Entomology. Blackwell Publishing Ltd., Oxford, U.K. Hall, T.A., 1999. BioEdit: An User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98. Hebert, P.D.N., Cywinska, A., Ball, S.L., DeWaard, J.R., 2003a. Biological identifications through DNA barcodes. Proc. R. Soc. Lond. B. Biol. Sci. 270 (1512), 313–321. https://doi.org/10.1098/rspb.2002.2218. Hebert, P.D.N., Ratnasingham, S., DeWaard, J.R., 2003b. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B. Biol. Sci. 270 (Suppl_1), S96–S99. https://doi.org/10.1098/rsbl.2003.0025. Hebert, P.D.N., Penton, E.H., Burns, J.M., Janzen, D.H., Hallwachs, W., 2004a. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc. Natl. Acad. Sci. U.S.A. 101 (41), 14812–14817. https://doi.org/10.1073/pnas.0406166101. Hebert, P.D.N., Stoeckle, M.Y., Zemlak, T.S., Francis, C.M., 2004b. Identification of Birds through DNA barcodes. PLOS Biol. 2 (10), e312. https://doi.org/10.1371/journal.pbio.0020312.
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Hirase, S., Takeshima, H., Nishida, M., Iwasaki, W., 2016. Parallel mitogenome sequencing alleviates random rooting effect in phylogeography. Genome Biol. Evol. 8 (4), 1267– 1278. https://doi.org/10.1093/gbe/evw063. Jia, K., Jia, T., 2016. Comparison of antidiuretic activity of Ootheca Mantidis before and after processing and its medicinal part against insufficiency of kidney-yang and diuresis rats. China Pharm. 27 (7), 879–881. Kim, H.Y., Lee, Y.J., Ahn, Y.M., Tan, R., Park, J.H., Choi, E.S., Lee, S.H., Lee, H.S., Kang, D.K., Lee, H.S., 2016. Effect of mantidis ootheca on the mechanism of vasorelaxation in thoracic aorta. FASEB J. 30 (1), 945.9. Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16 (2), 111–120. https://doi.org/10.1007/BF01731581. Korea Institute of Oriental Medicine, 2019. Defining Dictionary for Medicinal Herbs. Available from: http://boncho.kiom.re.kr/codex (accessed 4 April 2019). Kramer, K.J., 1973. Oothecal proteins of the oriental praying mantid Tenodera sinensis. Insect Biochem. 3 (11), 297–302. https://doi.org/10.1016/0020-1790(73)90060-7. McCune, B., Mefford, M.J., 2011. PC-ORD. Multivariate Analysis of Ecological Data. Version 6. MjM. Software, Gleneden Beach, Oregon, U.S.A. Ming-zhe, X.U., 2012. Anti-atherosclerotic activities of two compounds from Mantidis Ootheca. J. Anhui Agri. Sci. 32, 57. National Institute of Food and Drug Safety Evaluation, 2012. The Dispensatory on the Visual and Organoleptic Examination of Herbal Medicine. National Institute of Food and Drug Safety Evaluation. Cheongwon, Korea, pp. 341–342.
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Patel, S., 2018. Biological of Statilia maculata (Thunberg) (Insecta: Mantodea: Mantidae). J. Adv. Zool. 39 (1), 1–5. Rivera, J., Svenson, G.J., 2016. The Neotropical ‘polymorphic earless praying mantises’–Part I: molecular phylogeny and revised higher‐level systematics (Insecta: Mantodea, Acanthopoidea). Syst. Entomol. 41 (3), 607–649. https://doi.org/10.1111/syen.12178. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30 (12), 2725–2729. https://doi.org/10.1093/molbev/mst197. Tan, Z., Lei, Y., Zhang, B., Huang, L., 1997. Comparison of pharmacological studies on Ootheca Mantidis. China Journal of Chinese Materia Medica 22 (8), 496–499. The Korea Food and Drug Administration, 2014. The Korean Pharmacophoeia, eleventh ed, Volume 2. Korea Food and Drug Administration. Seoul, Korea, pp. 31–32. Wang, X., Hou, F.X., Wang, Y.X., Li, J.D., Yuan, Y., Peng, C., Guo, J.L., 2015. Identification of original species of Mantidis Oötheca (Sangpiaoxiao) based on DNA barcoding. China Journal of Chinese Materia Medica 40 (20), 3963–3966. Wen, L.L., Wan, D.G., Ren, Y., Li, J.D., Guo, J.L., 2013. Corresponding relationship between Mantis and Mantidis oötheca (Sangpiaoxiao). China Journal of Chinese Materia Medica 38 (7), 966–968. Ye, F., Lan, X.E., Zhu, W.B., You, P., 2016. Mitochondrial genomes of praying mantises (Dictyoptera, Mantodea): rearrangement, duplication, and reassignment of tRNA genes. Sci Rep. 6, 25634. https://doi.org/10.1038/srep25634. Zhang, H.L., Ye, F., 2017. Comparative mitogenomic analyses of praying mantises (Dictyoptera, Mantodea): origin and evolution of unusual intergenic gaps. Int. J. Biol. Sci. 13 (3), 367–382, https://doi.org/10.7150/ijbs.17035. 21
Mantidis Oötheca species identificaiton
Zhang, L.P., Yu, D.N., Storey, K.B., Cheng, H.Y., Zhang, J.Y., 2018. Higher tRNA gene duplication in mitogenomes of praying mantises (Dictyoptera, Mantodea) and the phylogeny within Mantodea. Int. J. Biol. Macromol. 111, 787–795. https://doi.org/10.1016/j.ijbiomac.2018.01.016. Zhao, Z., Xiao, P., Xiao, Y., Yuen, J.P., 2007. Quality assurance of Chinese herbal medicines (CHMs). J. Food Drug Anal. 15 (14), 337–346.
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Mantidis Oötheca species identificaiton
Figure captions
Fig. 1. Morphological characteristics of the ootheca used for measurements. (A) Tenodera angustipennis. (B) Hierodula patellifera. (C) Tenodera sinensis. AC: width of air-filled layer. DA: angle of distal end. LE: length of emergence area. LG, length. NE: number of egg chambers. TH, thickness. WD: width. WE: width of emergence area. Asterisks (*) indicate points of attachment to substrate. Scale bars = 1 cm.
Fig. 2. Stereomicroscope micrographs showing the ootheca morphology of four species. (A– C) Tenodera angustipennis. (D–F) Tenodera sinensis. (G–I) Hierodula patellifera. (J–L) Hierodula sp. A, D, G, J. Dorsal view. B, E, H, K. Lateral view. C, F, I, L. Cross-sectional shape. Scale bars = 1 cm.
Fig. 3. Stereomicroscope micrographs showing the emergence area and surface of ootheca. (A–B) Tenodera angustipennis. (C–D) Tenodera sinensis. (E–F) Hierodula patellifera. (G– H) Hierodula sp. A, C, E, G. The shape of emergence area. B, D, F, H. Surface pattern on lateral view. Scale bars = 1 mm.
Fig. 4. Principal component analysis (PCA) performed using the eleven quantitative ootheca morphological variables from four species. AC: width of air-filled layer. DA: angle of distal end. LE: length of emergence area. LE/LG. LG: length. NE: number of egg chambers. TH: thickness. WD: width. WE: width of emergence area. WE/WD. WG: weight. open triangle: Tenodera angustipennis (1) (2-19-0001). filled triangle: Tenodera angustipennis (2) (2-19-
23
Mantidis Oötheca species identificaiton
0002). filled circle: Tenodera sinensis (2-09-0025). filled square: Hierodula patellifera (2-180193A). open square: Hierodula sp. (2-18-0193B).
Fig. 5. Comparison of CO1 sequences of Tenodera angustipennis, Tenodera sinensis, Hierodula patellifera, and Hierodula sp.
Fig. 6. Phylogenetic tree of 63 samples of four species and related species using the neighbor-joining statistics with 1,000 bootstrap replicates based on CO1 sequences. ChP, Pharmacopoeia of the People’s Republic of China 2015 edition. KHP, The Korean Pharmacopoeia 11th edition, 2014.
24
Appendix. Oothecae medicinal materials information of accession number for each sample in this molecular study. Asterisked accession number are sequences obtained in this study. Speices
Sequence_ID
site
Voucher No
Accession No.
Tenodera angustipennis (1)
TA(1)-1
China
2-19-0001-1
MK829263*
Tenodera angustipennis (1)
TA(1)-2
China
2-19-0001-2
MK829264*
Tenodera angustipennis (1)
TA(1)-3
China
2-19-0001-3
MK829265*
Tenodera angustipennis (1)
TA(1)-4
China
2-19-0001-4
MK829266*
Tenodera angustipennis (1)
TA(1)-5
China
2-19-0001-5
MK829267*
Tenodera angustipennis (1)
TA(1)-6
China
2-19-0001-6
MK829268*
Tenodera angustipennis (1)
TA(1)-7
China
2-19-0001-7
MK829269*
Tenodera angustipennis (1)
TA(1)-8
China
2-19-0001-8
MK829270*
Tenodera angustipennis (1)
TA(1)-9
China
2-19-0001-9
MK829271*
Tenodera angustipennis (1)
TA(1)-10
China
2-19-0001-10
MK829272*
Tenodera angustipennis (1)
TA(1)-11
China
2-19-0001-11
MK829273*
Tenodera angustipennis (1)
TA(1)-12
China
2-19-0001-12
MK829274*
Tenodera angustipennis (1)
TA(1)-13
China
2-19-0001-13
MK829275*
Tenodera angustipennis (1)
TA(1)-14
China
2-19-0001-14
MK829276*
Tenodera angustipennis (2)
TA(2)-1
China
2-19-0002-1
MK829277*
Tenodera angustipennis (2)
TA(2)-2
China
2-19-0002-2
MK829278*
Tenodera angustipennis (2)
TA(2)-3
China
2-19-0002-3
MK829279*
Tenodera angustipennis (2)
TA(2)-4
China
2-19-0002-4
MK829280*
Tenodera angustipennis (2)
TA(2)-5
China
2-19-0002-5
MK829281*
Tenodera angustipennis (2)
TA(2)-6
China
2-19-0002-6
MK829282*
Tenodera angustipennis (2)
TA(2)-7
China
2-19-0002-7
MK829283*
Tenodera angustipennis (2)
TA(2)-8
China
2-19-0002-8
MK829284*
Tenodera angustipennis (2)
TA(2)-9
China
2-19-0002-9
MK829285*
Tenodera angustipennis (2)
TA(2)-10
China
2-19-0002-10
MK829286*
Tenodera angustipennis (2)
TA(2)-11
China
2-19-0002-11
MK829287*
Tenodera angustipennis (2)
TA(2)-12
China
2-19-0002-12
MK829288*
Tenodera angustipennis (2)
TA(2)-13
China
2-19-0002-13
MK829289*
Tenodera angustipennis (2)
TA(2)-14
China
2-19-0002-14
MK829290*
Tenodera angustipennis (2)
TA(2)-15
China
2-19-0002-15
MK829291*
Tenodera angustipennis
C3
China
-
KR052038
Tenodera sinensis Tenodera sinensis
TS-1
China
2-09-0025-1
MK829292*
TS-2
China
2-09-0025-2
MK829293*
Tenodera sinensis
TS-3
China
2-09-0025-3
MK829294*
Tenodera sinensis
TS-4
China
2-09-0025-4
MK829295*
Tenodera sinensis
TS-5
China
2-09-0025-5
MK829296*
Tenodera sinensis
TS-6
China
2-09-0025-6
MK829297*
Tenodera sinensis
TS-7
China
2-09-0025-7
MK829298*
Tenodera sinensis
TS-8
China
2-09-0025-8
MK829299*
Tenodera sinensis
-
China
-
KY689132
Hierodula formosana
-
Taiwan
Jftl-1
KR703238
Hierodula patellifera
HP-1
China
2-19-0003A-1
MK829300*
Hierodula patellifera
HP-2
China
2-19-0003A-2
MK829301*
a
b c
Hierodula patellifera
HP-3
China
2-19-0003A-3
MK829302*
Hierodula patellifera
HP-4
China
2-19-0003A-4
MK829303*
Hierodula patellifera
HP-5
China
2-19-0003A-5
MK829304*
Hierodula patellifera
HP-6
China
2-19-0003A-6
MK829305*
Hierodula patellifera
HP-7
China
2-19-0003A-7
MK829306*
Hierodula patellifera
HP-8
China
2-19-0003A-8
MK829307*
Hierodula patellifera
HP-9
China
2-19-0003A-9
MK829308*
Hierodula patellifera
HP-10
China
2-19-0003A-10
MK829309*
Hierodula patellifera
HP-11
China
2-19-0003A-11
MK829310*
Hierodula patellifera
-
China
-
KX611803
Hierodula sp.
HS-1
China
2-19-0003B-1
MK829311*
Hierodula sp.
HS-2
China
2-19-0003B-2
MK829312*
Hierodula sp.
HS-3
China
2-19-0003B-3
MK829313*
Hierodula sp.
HS-4
China
2-19-0003B-4
MK829314*
Hierodula sp.
HS-5
China
2-19-0003B-5
MK829315*
Hierodula sp.
HS-6
China
2-19-0003B-6
MK829316*
Hierodula sp.
HS-7
China
2-19-0003B-7
MK829317*
Hierodula sp.
HS-8
China
2-19-0003B-8
MK829318*
Hierodula sp.
HS-9
China
2-19-0003B-9
MK829319*
Hierodula sp.
HS-10
China
2-19-0003B-10
MK829320*
Hierodula sp.
HS-11
China
2-19-0003B-11
MK829321*
Hierodula sp.
HS-12
China
2-19-0003B-12
MK829322*
Hierodula sp.
H4
China
-
KR011164
Mantis religiosa
-
-
-
FJ802846
d
a
Statilia apicalis
-
-
-
KR011166
Statilia maculata
-
-
-
KT036560
-
-
NC014887
† a
-
Acrida cinerea b
c
d
†, outgroup. Wang et al. (2015). Zhang et al. (2018). Tian et al. (2015). Zhang and Ye (2017).
Table 1. Overview of representative morphological characteristics of oothecae in four Mantis species.
Characters
Tenodera angustipennis (1)
Tenodera angustipennis (2)
Tenodera sinensis
Hierodula patellifera
Hierodula sp.
Shape, outline
fu
fu
ba
el
el
obo
obo
cir to orb
cir
cir
D br/du
D br/du
L br to Y br/sh
R D br to R br/sh
R D br to D br/sh
Length (LG, mm)
34.37 ± 2.96
33.3 ± 4.19
27.9 ± 3.98
26.87 ± 2.93
25.44 ± 2.53
Width (WD, mm)
9.80 ± 0.50
9.87 ± 0.93
23.7 ± 1.97
12.05 ± 1.01
13.3 ± 0.59
Thickness (TH, mm)
9.15 ± 0.91
9.03 ± 0.98
19.1 ± 3.42
10.96 ± 0.84
14.1 ± 0.61
Length of emergence area (LE, mm)
25.14 ± 2.42
25.13 ± 3.44
22.0 ± 3.41
19.04 ± 2.95
22.7 ± 2.46
Width of emergence area (WE, mm)
3.92 ± 0.56
4.11 ± 0.62
6.11 ± 1.01
4.24 ± 0.51
4.23 ± 0.32
fl
fl
fl
fl
so
Number of egg chambers (NE, no)
24.6 ± 2.68
24.1 ± 3.66
20.9 ± 2.98
17.9 ± 1.92
16.9 ± 2.16
Angle of distal end (DA, ˚)
38.8 ± 5.99
39.9 ± 6.82
85.9 ± 8.57
61.3 ± 5.09
91.7 ± 5.14
Width of air-filled layer (AC, mm)
0.69 ± 0.19
0.63 ± 0.10
6.62 ± 1.32
1.09 ± 0.31
0.62 ± 0.16
0.452 ± 0.081
0.418 ± 0.124
0.827 ± 0.17
0.58 ± 0.11
1.04 ± 0.17
Cross-sectional shape Color/shiny
Opening of egg chamber
Weight (WG, g)
Note: All quantitative characters show (abbreviation, unit). Numbers refer to means ± S.D. (sample size n = 100). Shape: ba, barrel-like; el, ellipsoid; fu, fusiform. Cross-sectional shape: cir, circular; obo, obovate; orb, orbicular. Color/shiny: br, brown; D, dark; du, lusterless; L, light; R, reddish; sh, lustrous; Y, yellowish. Opening of egg chamber: fl, flexible flap; so, single opening.
Table 2. The results of the principal component analysis of the eleven quantitative oothecae morphological characters of four species from medicinal materials. PC 1
PC 2
PC 3
(49.884%)
(20.271%)
(14.288%)
No.
Characteristics
1
LG
0.6554
-0.6541
-0.0187
2
WD
-0.8794
-0.3881
-0.2071
3
TH
-0.927
-0.2365
-0.0357
4
LE
0.3627
-0.6812
0.6064
5
WE
-0.5969
-0.5881
-0.1919
6
LE/LG
-0.3998
-0.0018
0.8353
7
WE/WD
0.7811
-0.0827
0.0485
8
NE
0.5377
-0.7174
0.0434
9
DA
-0.9179
0.1571
0.2667
10
AC
-0.7193
-0.4891
-0.4041
11
WG
-0.7198
-0.0085
0.4315
5.487
2.230
1.572
Eigenvalue
Note: The first three PCs with eigenvalue larger than one were represented. The percentages in parentheses indicate the amount of variation explained by each PC, and the components that were loaded most highly for each character are in bold. Total explanation power 84.443 %.
Table 3. Statistical characteristics of CO1 regions among studied species. Species Tenodera angustipennis Tenodera sinensis Hierodula patellifera Hierodula sp.
Amplicon Length (bp) 694 694 694 694
Intra-Species Distance (%) 0.16 ± 0.15 0.41 ± 0.22 0.20 ± 0.14 0.10 ± 0.09
G+C (%) 33.32 31.55 32.73 32.78
Table 4. Nucleotide sequence distance matrix generated by the Kimura-2-parameter (K2P) distance model among all studied species except outgroup, based on the CO1 mitochondrial gene. S. apicalis
S. maculata
-
-
-
-
-
-
-
-
-
-
-
-
13.67
-
-
-
-
-
14.47
13.37
1.93
-
-
-
-
16.25
14.04
17.12
15.46
15.56
-
-
-
S. apicalis
17.91
14.84
16.89
16.75
16.61
13.78
-
-
S. maculata
18.12
15.05
16.68
16.96
16.82
13.98
0.50
-
(%)
Hierodula sp. H. formosana M. religiosa
T. angustipennis
T. sinensis
H. patellifera
-
-
-
-
-
7.22
-
-
-
H. patellifera
14.90
13.43
-
Hierodula sp.
17.67
14.98
H. formosana
15.86
M. religiosa
T. angustipennis T. sinensis
S0378-8741(19)32336-0
DOI:
https://doi.org/10.1016/j.jep.2020.112574
Reference:
JEP 112574
To appear in:
Journal of Ethnopharmacology
Received Date: 12 June 2019 Revised Date:
3 December 2019
Accepted Date: 13 January 2020
Please cite this article as: Song, J.-H., Cha, J.-M., Moon, B.C., Kim, W.J., Yang, S., Choi, G., Mantidis Oötheca (mantis egg case) original species identification via morphological analysis and DNA barcoding, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/j.jep.2020.112574. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Mantidis Oötheca species identificaiton
(Original research articles)
Mantidis Oötheca (mantis egg case) original species identification via morphological analysis and DNA barcoding
Jun-Ho Song, Ji-Min Cha, Byeong Cheol Moon, Wook Jin Kim, Sungyu Yang, Goya Choi*
Herbal Medicine Reseources Research Center, Korea Institute of Oriental Medicine, Naju, 58245, Republic of Korea
*Corresponding author: Telephone: +82-61-338-7118 Fax: +82-61-338-7135
E-mail addresses: [email protected] (J.-H. Song), [email protected] (J.-M. Cha), [email protected] (B. C. Moon), [email protected] (W. J. Kim), [email protected] (S. Yang), [email protected] (G. Choi).
Running title: Mantidis Oötheca species identification
Number of pages: 24 Number of figures: 6 Number of tables: 4 1
Mantidis Oötheca species identificaiton
Abstract Ethnopharmacological relevance: Mantidis Oötheca (mantis egg case; sangpiaoxiao) is a medicine from an insect source, which has been widely used in Asian countries. However, misidentification due to a lack of information given variations in the medicinal portion of the ootheca and morphological similarities of the ootheca as an egg chamber. Aim of the study: Thus, this study aims to provide the first comprehensive data for discriminating authentic of Mantidis Oötheca. Here, we provide detailed ootheca morphology and their molecular information to accurately identify Mantidis Oötheca. Materials and methods: Oothecae of Tenodera angustipennis (Saussure, 1869), Tenodera sinensis (Saussure, 1871), Hierodula patellifera Serville, 1839, and Hierodula sp. were used in the comparative morphological, principal component analysis, and DNA barcoding. Results: The morphological analyses revealed that the emergence area, outline, angle of distal end, width of air-filled layer, and weight are useful diagnostic characters. Using these quantitative and qualitative characteristics, we developed the effective identification key. Furthermore, our CO1 sequences from all individuals were monophyletic with high bootstrap values at genus and species levels. Moreover, morphological identification using our developed key among all studied individuals agreed with molecular identification results using CO1 barcoding data. Conclusions: These multilateral approaches, including morphological, statistical, and DNA barcoding methods are highly reliable identification tools. Moreover, our diagnostic key characteristics and molecular barcoding should aid in the accurate identification, authentication, and quality control of Mantidis Oötheca medicinal materials.
Keywords: Mantidis Oötheca, Tenodera, Hierodula, PCA, CO1 barcoding, identification. 2
Mantidis Oötheca species identificaiton
1. Introduction As a group, insects comprise about 70% of all living organisms (Gullan and Cranston, 2005). Insects and insect-derived products have been used as sources of food, medicine, and chemical materials for a long time on account of their great diversity and richness. In particular, medicinal insects and their derived products are used to treat various diseases, a practice called entomotherapy (Costa-Neto, 2005; Fan and Ding, 2001; Feng et al., 2009). Insect bodies, eggs, egg shells, exuviae, and secretions are still used in traditional medicine (Feng et al., 2009). Traditional medical practices in Asian countries, including Korea, China, and Taiwan, commonly use mantis ootheca (i.e., an egg shell, egg chamber, or egg case), which is a complex structure that female praying mantises provide to support and protect their eggs from adverse environmental conditions and natural predators (Kramer, 1973). According to the Korean Herbal Pharmacopoeia, authentic Mantidis Oötheca refers to only the steamed ootheca of Tenodera angustipennis (Saussure, 1869) (tribe Polyspilotini), Statilia maculata (Thunberg, 1784) (tribe Mantini), or Hierodula patellifera Serville, 1839 (tribe Paramantini), all of which belongs to the Mantidae family (Korea Food and Drug Administration, 2014; Korea Institute of Oriental Medicine, 2019). The Pharmacopoeia of the People’s Republic of China and the Taiwan Herbal Pharmacopoeia have designated the ootheca of Tenodera sinensis (Saussure, 1871) (tribe Polyspilotini), Statilia maculata (Thunberg, 1784), and Hierodula patellifera Serville, 1839 as authentic Mantidis Oötheca (Sangpiaoxiao), called "Tuanpiaoxiao", "Changpiaoxiao", or "Heipiaoxiao", respectively (Chinese Pharmacopoeia Commission, 2015; Committee on Chinese Medicine and Pharmacy, 2013; Korea Institute of Oriental Medicine, 2019). The differences between the authentic species, which are designated in the Pharmacopoeias of each country, result in the 3
Mantidis Oötheca species identificaiton
distribution of adulterants or counterfeits in markets (National Institute of Food and Drug Safety Evaluation, 2012). Traditionally, Mantidis Oötheca have been used for the clinical treatment of incontinence and frequent urination (Korea Institute of Oriental Medicine, 2019). Modern pharmacological studies have shown that this medicinal insect has anti-diuretic (Jia and Jia, 2016; Tan et al., 1997), anti-oxidant, anti-atherosclerotic (Ming-zhe, 2012), and vasorelaxation (Kim et al., 2016) properties. However, a number of reports have emerged regarding the medicinal portion of the ootheca that indicate misidentifications due to the great variety of mantis ootheca available that are morphologically similar (National Institute of Food and Drug Safety Evaluation, 2012; Wen et al., 2013). Moreover, mixed oothecae from different species within one package are currently sold in commercial markets. Thus, it is necessary to provide accurate information regarding the species present in Mantidis Oötheca in order to ensure quality. Previous studies on the relationship between Mantis, Mantidis Oötheca (Wen et al., 2013), and identification based on DNA barcoding (Wang et al., 2015) have been conducted. However, oothecae morphology, which contains great potential for taxonomic and systematic classification, has not yet been studied. Therefore, we carried out this study to (1) provide detailed descriptions and illustrations of the morphology of Mantidis Oötheca, including adulterants, to create a morphological identification key, (2) to determine phylogenetic relationships using universal DNA barcode sequences, and (3) to evaluate the relationship between morphology and molecular data for accurate identification.
2. Materials and Methods 4
Mantidis Oötheca species identificaiton
2.1. Medicinal Materials Oothecae of Tenodera angustipennis and T. sinensis (Tuanpiaoxiao), which have been designated as authentic herbal materials in the Korean Herbal Pharmacopoeia (KHP) and the Pharmacopoeia of the People’s Republic of China (ChP), respectively, were used in the analysis. The oothecae of Hierodula patellifera (Heipiaoxiao), which is described as Mantidis Oötheca both the KHP and ChP, and Hierodula sp., which is currently mixed with adulterant species in medicinal markets due to morphological similarities, were also included. Ootheca terminology and identification follows Breland and Dobson (1947), Chinese Medicinal Material Images Database (2012), and Brannoch et al. (2017). All medicinal materials were purchased in medicinal markets from commercial suppliers and samples were deposited in the Korean Herbarium of Standard Herbal Resources (Index Herbariorum code KIOM) at the Korea Institute of Oriental Medicine, Naju, Korea (T. angustipennis, 2-19-0001, 2-19-0002; T. sinensis, 2-09-0025; H. patellifera, 2-19-0003A; and Hierodula sp., 2-19-0003B.
2.2. Morphological Analysis Measurements and optical observations of 20 randomly selected oothecae from each of the five vouchers were recorded for a total of 100 oothecae. A set of digital Vernier calipers (CD-15CP, Mitutoyo, Kawasaki, Japan) was used to measure the length (LG), width (WD), thickness (TH), length of emergence area (LE), width of emergence area (WE), and width of air-filled layer (AC; Fig. 1). Furthermore, a digital protractor was used to measure angle of distal end (DA). The general shape, cross-sectional shape, color, the number of egg chambers (NE), emergence area, and the flaps were recorded using an optical and a stereomicroscope (Olympus SZX16, Olympus, Tokyo, Japan). Ootheca cross-sections were prepared by cutting down the middle of the ootheca using a single-edge blade (DN-52, 5
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Dorco, Seoul, Korea), and images were captured using a digital camera. The weight of the ootheca (WG) was measured using an electronic precision balance (PS 1000.R1, RADWAG Balances & Scales, Radom, Poland).
2.3. Principal Component Analysis A principal component analysis (PCA) was performed to verify whether the quantitative morphological data allowed species grouping. The first three principal components (PCs) with eigenvalues larger than one were represented. The results were presented in a two-dimensional plot of the first and second principal components. These analyses were performed using the program PC-ORD version 5.31 (McCune and Mefford, 2011). The PCA analysis was performed with eleven variables (LG, WD, TH, LE, WE, LE/LG, WE/WD, NE, DA, AC, and WG).
2.4. Preparation of Genomic DNA All medicinal materials were identified based on morphological characteristics and total genomic DNA was extracted from 60 individuals (Appendix). DNA was extracted using a DNeasy® Blood and Tissue Kit (Qiagen, Valencia, CA, USA) according to the protocol. The purity and concentration of extracted DNA were assessed using a spectrophotometer (Nanodrop ND-1000, Nanodrop, Wilmington, DE, USA) and 1.5% agarose gel electrophoresis. The final DNA concentration used for PCR amplification was approximately 15 ng/µL in TE buffer. Extracted DNA samples were stored at -20°C (Kim et al., 2016).
2.5. PCR Amplification of CO1
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Using the primers (CO1-C02 5′-AYT CAA CAA ATC ATA AAG ATA TTG G-3′ and CO1-C04 5′-ACY TCR GGR TGA CCA AAA AAT CA-3′) developed by Che et al. (2012) a fragment of the mitochondrial cytochrome oxidase subunit 1 (CO1) was amplified. PCR amplifications were performed in 40 µL reaction volumes containing 10 mmol/L TrisHCl (pH 9.0), 2.5 mmol/L MgCl2, 200 µmol/L each dNTP, 10 mmol/L (NH4)2SO4, 0.5 U Taq DNA polymerase (Solgent, Daejeon, Korea), 0.5 µmol/L each primer, and approximately 15 ng of template DNA (Kim et al., 2016). PCR amplification was performed using a DNA Engine Dyad® PTC-0220 (Bio-Rad, Foster City, CA, USA). The modified parameters were as follows: 95°C for 5 min; 35 cycles of 1 min at 95°C, 1 min at 45°C, and 1 min at 72°C; and a final extension for 5 min at 72°C (Kim et al., 2016). PCR products were separated using 1.5% agarose gel electrophoresis with a 100-bp DNA ladder (Solgent, Daejeon, Korea).
2.6. Nucleotide Sequence and Phylogenetic Analysis Amplified mitochondrial CO1 DNA fragments were rescued from agarose gels using a QIAquick® Gel Extraction Kit (Qiagen, Valencia, CA, USA) and subcloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). Inserted fragments were sequenced in both directions using an automatic DNA sequence analyzer (ABI 3730, Applied Biosystems Inc., Foster City, CA, USA; Kim et al., 2016). The samples of Hierodula patellifera (11 individuals; abbreviation HP), Hierodula sp. (12 individuals; HS), Tenodera angustipennis (29 individuals; TA), and T. sinensis (8 individuals; TS) were used in the analysis (Appendix). To construct the phylogenetic tree, CO1 sequences of eight related species were downloaded from NCBI GenBank: H. formosana (Giglio-Tos, 1912), KR703238; H. patellifera, KX611803; Hierodula sp., KR011164; Mantis religiosa (Linnaeus, 1758), FJ802846; Statilia 7
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apicalis (Saussure, 1871), KR011166; S. maculata, KT036560; T. angustipennis, KR052038; T. sinensis, KY689132. The 694-bp CO1 sequences were assembled and edited using BioEdit version 7.2.5 (Hall, 1999). The contigs were aligned to analyze intra- and inter-species variations in the sequences. For distance analyses, pairwise sequence divergences were calculated using the Kimura-2-parameter (K2P) distance model in MEGA 6 software. The phylogenetic analysis based on the entire CO1 sequences was performed by MEGA 6 version 6.06 (Kimura, 1980; Tamura et al., 2013). The phylogenetic tree was constructed using the NJ method with the K2P model as recommended by Hebert et al. (2003a), the pairwise deletion for gaps/missing data treatment, and 1000 replications for bootstrapping with Acrida cinerea (Thunberg, 1815) (NC014887) as an outgroup.
3. Results 3.1. Morphological Characteristics The morphological variation of the ootheca of four species is described. The representative and quantitative ootheca characteristics of all species evaluated are summarized in Table 1. Representative oothecae of the four species are illustrated in Figs. 1– 3. The oothecae of studied species were fusiform (Fig. 2A), barrel-like (Fig. 2D), and ellipsoid (Fig. 2G, J) in the dorsal view, and obovate (Fig. 2C) and mostly circular to orbicular (Fig. 2F, I, L) the cross- sectional view. The proximal end of all studied ootheca was either partially or fully encircling the substrate to which it was attached (usually a branch, branchlet, or stem) from the proximal to nearly the distal end in such a way that the ootheca laid with its ventral surface hidden, and the emergence area was almost parallel to the 8
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substrate. Tenodera sinensis possessed the largest ootheca (27.9 × 23.7 mm average area; Fig, 2D–E), whereas T. angustipennis had the smallest ootheca among species studied (33.84 × 9.84 mm; Fig. 2A–B). The external wall color ranged from light brown to yellowish brown (Fig. 2D–E) or reddish dark brown to dark brown (Fig. 2A–B, G–H, J–K). In addition, the external wall was either lustrous (Fig. 2G–L) or lusterless (Fig. 2A–F), soft or hard in strength, and smooth or sponge-like in texture. The studied oothecae contained 13–33 egg chambers that were aligned one after another in zigzag arrangement (Fig. 3A, C, E, G). The emergence area exhibited a single emergence opening (Fig. 3G) or a flexible flap (Fig. 3A, C, E).
3.2. Description of Four Species Ootheca Tenodera angustipennis (Sangpiaoxiao). Ootheca fusiform (i.e., proximal end obtuse, distal end tapered; Fig. 2A, B) and mostly obovate in cross-section (Fig. 2C). Ventral part of ootheca formed a furrow from the proximal to distal end (Fig. 2C). External wall dark brown in color, somewhat lusterless and somewhat hard in strength. External coating is a brownish textured. Exhibiting 19–33 egg chambers whose boundaries are clearly delimited, visible on the lateral view as parallel ridges that obliquely arrange on the ventral furrow (Fig. 3B). Distal end of ootheca tapered, dorsoventrally compressed and slightly inclined in the lateral view (Fig. 2B). Emergence area composed of multiple openings, significantly elevated, and all aligned to form a zigzag arranged in two parallel rows and along the dorsal longitudinal axis of the ootheca. Each egg chamber exhibits a flexible flap (i.e., an operculum) that partially closes its corresponding opening. These flaps project slightly beyond the edge of the ootheca; the apex of flaps is rounded (Fig. 3A). Measurements (Table 1): length, 23.81–40.44 mm; width, 7.80–11.54 mm; thickness, 7.50–11.96 mm; length of emergence area, 18.10–30.30 mm; width of emergence area, 3.07– 9
Mantidis Oötheca species identificaiton
5.47 mm; number of egg chambers, 19–33; angle of distal end, 22.1–50.1°; width of air-filled layer, 0.29–1.03 mm; weight, 0.16–0.62 g.
Tenodera sinensis (Tuanpiaoxiao). Ootheca barrel-like (Fig. 2D, E) and mostly circular to orbicular in cross-section (Fig. 2F). Ventral part of ootheca formed a wide furrow from the proximal to distal end (Fig. 2F). External wall light brown to yellowish brown in color, somewhat lustrous, soft in strength, and sponge-like in texture. Exhibiting 16–26 egg chambers whose boundaries are indistinct, visible on the lateral view as parallel ridges that almost perpendicularly arrange to the ventral furrow (Fig. 3D). Distal end of ootheca truncated, rough, and steep in incline in the lateral view (Fig. 2E). Emergence area composed of multiple openings, all aligned to form a nearly zigzag pattern, arranged in two parallel rows along the dorsal longitudinal axis of the ootheca. Each egg chamber exhibits a flexible flap that completely closes its corresponding opening. These flaps project slightly beyond the edge of the ootheca, and the apex of flaps is rounded or obtuse (Fig. 3C). Measurements (Table 1): length, 19.62–34.03 mm; width, 20.19–27.37 mm; thickness, 13.14–25.26 mm; length of emergence area, 16.20–31.40 mm; width of emergence area, 4.60–8.98 mm; number of egg chambers, 16–26; angle of distal end, 63.6–98.7°; width of air-filled layer, 3.69–8.59 mm; weight, 0.49–1.14 g.
Hierodula patellifera (Heipiaoxiao). Ootheca ellipsoid (Fig. 2G) and mostly circular in cross-section (Fig. 2I). Ventral part of ootheca formed furrow from the proximal to distal end (Fig. 2I). External wall reddish dark brown to reddish brown in color, lustrous, hard in strength, and smooth. Exhibiting 14–21 egg chambers whose boundaries are clearly delimited, visible in the lateral view as parallel ridges that are obliquely arranged on the ventral furrow (Fig. 3F). Distal end of ootheca slightly tapered, with a moderate incline in the 10
Mantidis Oötheca species identificaiton
lateral view. Emergence area composed of multiple openings, all aligned to form a zigzag arranged in two parallel rows along the dorsal longitudinal axis of the ootheca. Each egg chamber exhibits a flexible flap that completely closes its corresponding opening. These flaps project beyond the edge of the ootheca, the apex of flaps is obtuse (Fig. 3E). The flaps of the distal end of ootheca are situated upwards (Fig. 2H). Measurements (Table 1): length, 20.23–30.26 mm; width, 10.23–14.04 mm; thickness, 9.26–12.5 mm; length of emergence area, 12.05–23.67 mm; width of emergence area, 3.40–5.42 mm; number of egg chambers, 14–21; angle of distal end, 52.8–69.6°; width of air-filled layer, 0.25–1.63 mm; weight, 0.41–0.73 g.
Hierodula sp. Ootheca ellipsoid (Fig. 2J) and mostly circular in cross-section (Fig. 2L). Ventral part of ootheca formed furrow from the proximal to almost the distal end (Fig. 2L). External wall reddish dark brown to dark brown in color, lustrous, very hard in strength, and smooth. Exhibiting 13–21 egg chambers whose boundaries are clearly delimited, visible in the lateral view as parallel ridges that are obliquely arranged on the ventral furrow (Fig. 3H). Distal end of ootheca truncated, rough, with a steep incline in the lateral view (Fig. 2K). Emergence area slightly elevated and composed of approximately 25–42 openings, all aligned to form a zigzag arranged in two parallel rows along the dorsal longitudinal axis of the ootheca (Fig. 3G). Measurements (Table 1): length, 20.95–29.93 mm; width, 11.60–14.09 mm; thickness, 12.57–15.14 mm; length of emergence area, 17.95–27.40 mm; width of emergence area, 3.56–4.94 mm; number of egg chambers, 13–21; angle of distal end, 79.3–99.2°; width of air-filled layer, 0.42–0.94 mm; weight, 0.81–1.35 g.
3.3. Principal Component Analysis (PCA) 11
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The relationships among the species evaluated with quantitative ootheca morphological data were explored using a PCA analysis (Fig. 4). The first two PCs explained 70.16 % of the total variance of the analyzed data (Table 2). The first principal component (PC 1) accounted for 49.88 % of the variance based on the values of the ootheca size (LG, WD, TH, DA), emergence area size (WE, WE/WD), ootheca shape (DA), and weight (WG; Table 2). The second principal component (PC 2) explained 20.27 % of the data variability, of which length of emergence area (LE) and the number of egg chambers (NE) were the significant variables for the ordination of species (Table 2). The PCA biplot reveals the groupings of the operational taxonomic units (OTUs) that correspond to each species. For the species T. angustipennis, the same species OTUs but different vouchers, are grouped on the positive side of the PC 1 axis, while all OTUs of T. sinensis are grouped on the negative side of the PC 1 axis (Fig. 4). Most of the OTUs of both H. patellifera and Hierodula sp. are positioned on the positive side of the PC 2 axis. However, OTUs of H. patellifera are positioned on the central to positive side of the PC 1 axis, while and Hierodula sp. OTUs are positioned on the positive side of the PC 1 axis with regard to the data from DA (Fig. 4).
3.4. Identification Key Based on Ootheca Morphology 1. Ootheca barrel-like in shape; external wall light brown to yellowish brown in color, with sponge-like texture; width of air-filled layer, 3.69–8.59 mm ------------------------------------------------------------------------------------------------------------ Tenodera sinensis (Saussure, 1871) – Ootheca fusiform or ellipsoid in shape; external wall reddish dark brown to reddish brown in color, and not sponge-like in texture; width of air-filled layer, 0.25–1.63 mm -------------- 2 2. Ootheca fusiform in shape (distal end tapered); external wall lusterless, brownish textured; angle of distal end 22.1–50.1° -------- Tenodera angustipennis (Saussure, 1869)
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– Ootheca ellipsoid in shape (distal end not tapered); external wall lustrous, smooth; angle of distal end 52.8–99.2° --------------------------------------------------------------------------- 3 3. External wall hard in strength; each egg chamber exhibits a flexible flap; angle of distal end 52.8–69.6°; weight 0.41–0.73 g ---------- Hierodula patellifera Serville, 1839 – External wall very hard in strength; each egg chamber exhibits a single emergence opening; angle of distal end 79.3–99.2°; weight 0.81–1.35 g ---------------- Hierodula sp.
3.5. Genetic Variability and Phylogenetic Relationships The length of the CO1 region was 694-bp in over 60 of the ootheca studied (Table 3, Fig 5, see Appendix for accession numbers). The intra-specific genetic sequence variability was 0.10% for Hierodula sp., 0.16% for Tenodera angustipennis, 0.20% for H. patellifera, and 0.41% for T. sinensis (Table 3). Inter-specific genetic distance values ranged from 0.50 (Statilia maculata-S. apicalis) to 18.12% (T. angustipennis-S. maculata; Table 4). The interspecific genetic distances of each congeneric species were 0.50% in Statilia and 7.22% in Tenodera. Hierodula sp. is genetically closer to H. formosana (1.93%) than H. patellifera (13.67%) (Table 4). The phylogenetic tree constructed using the neighbor-joining (NJ) method revealed that all samples (operational taxonomic units) were clustered monophyletically with high bootstrap values at the species level (100%; Fig. 6). Within the studied species, this clade is divided into two distinct and well-supported; one of the clades contained T. angustipennis and T. sinensis, while the other contained H. patellifera, H. formosana, and Hierodula sp. (Fig. 6). Within the genus Hierodula, H. formosana were closely related to Hierodula sp. (levels of a genetic distance value, 1.93%; Table 4, Fig. 6).
4. Discussion
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The ootheca characteristics of the species evaluated show diversity in general shape, texture, strength, the width of the air-filled layer of the external wall, and the type of emergence opening present (Table 1). Although mantis oothecae have been used for important medicinal purposes as Mantidis Oötheca, their macroscopic structure remains largely understudied. Moreover, misidentification and incorrect information exists with regard to this product (National Institute of Food and Drug Safety Evaluation, 2012). Misuse of traditional medicines due to adulterants or counterfeits may even result in severe harm to patients (Zhao et al., 2007). Thus, the accurate identification of authentic Mantidis Oötheca is necessary to safeguard their clinical effects. Morphological characteristics of ootheca were first used in taxonomic studies (Breland and Dobson, 1947). Since the time of Breland and Dobson (1947), ootheca characteristics have been currently used for classifying taxa (Brannoch et al., 2017; Rivera and Svenson, 2016). In particular, Brannoch et al. (2017) suggest that the following relevant diagnostic features should be included in species descriptions: shape, size, external wall, point of attachment, egg chambers, and emergence area. The morphological variation of ootheca from this study agrees with those of previously published data from specimens collected from America (Breland and Dobson, 1947). The emergence area of T. angustipennis, with a significantly elevated ridge (Fig. 2B–C), resembles that of the American oothecae of the same species (Breland and Dobson, 1947, Plate V, Fig. 2). Moreover, oothecae of T. sinensis from America (Breland and Dobson, 1947, Plate VI, Fig. 1), which are characterized by larger ootheca mass, globular outline (i.e., barrel-like), and evident air spaces (i.e., width of air-filled layer), were also similar to those of the present study (Fig. 2D–F). Thus, these retained morphological characteristics, which show no geographical variation, could represent important and stable diagnostic keys. Oothecae of Hierodula sp., which are morphologically similar to those of an authentic species H. patellifera, showed single 14
Mantidis Oötheca species identificaiton
emergence opening (Fig. 3G) with a distal angle of approximately 80–100° (Fig. 2K) and were more than 0.8 g per sample heavier than those of H. patellifera. These data suggest that the presence of flexible flap, the angle of distal end, and weight per ootheca are convenient and useful characteristics that can be used for the immediate identification of medicinal insect ootheca. Moreover, our PCA analysis based on the data matrix revealed that the OTUs of each species were well-clustered (Fig. 4). This data matrix, which includes the useful quantitative data of correctly identified species, gives us objective evidences to identify the oothecae. Thus, our data matrix and statistic techniques might be effective tools for more accurate identification of medicinal ootheca. Oothecae of S. maculata, known as a "Changpiaoxiao", has been listed in the Pharmacopoeia of Asian countries including Korea, China, and Taiwan (Korea Institute of Oriental Medicine, 2019). However, we found that the ootheca of S. maculata was not distributed in Korean pharmaceutical and herbal medicinal markets through our market survey. Moreover, morphological characteristics such as shape and color of the oothecae, differ from our studied oothecae (Patel, 2018). The mitochondrial genome is a powerful molecular marker (Boore, 2006; Hirase et al., 2016). In particular, the cytochrome c oxidase 1 (CO1) sequence was sufficient to generate DNA barcodes for species identification (Hebert et al., 2003a, b, 2004a, b). In order to evaluate the origin and evolution of Mantodea, phylogenetic studies using mitochondrial genomes have recently been conducted (Ye et al., 2016; Zhang and Ye, 2017; Zhang et al., 2018). In addition, molecular identification using the CO1 sequence has also been evaluated (Wang et al., 2015). Our sequence data from all individuals of each species were monophyletic with high bootstrap values at the genus and species levels (Fig. 6). This kind of phylogenetic tree agrees 15
Mantidis Oötheca species identificaiton
with previous results (Wang et al., 2015). Moreover, the morphological identification among all individuals studied corresponds perfectly with the molecular identification results using COI barcoding data. Interestingly, we found that two morphologically different oothecae are mixed together and sold in medicinal markets. Based on the presence of a flexible flap, the angle of distal end, and weight per ootheca, one was identified as a H. patellifera and the other was identified as Hierodula sp. According to the Wang et al. (2015) phylogeny, clade 3 (H1~H5), which is monophyletic, verifies Hierodula sp. not H. patellifera; although they did not recognize morphological differences with the oothecae of H. patellifera, oothecae were divided into two branches based on CO1 (Wang et al., 2015). Our sequence data of Hierodula sp., which have single emergence opening egg chamber, a 79.3–99.2° angle of the distal end, and are 0.81–1.35 g, are identical with Hierodula sp. from Wang et al. (2015). Moreover, we found that Hierodula sp. is more closely related to H. formosana than H. patellifera. Thus, using these morphological and molecular data, further study is required to accurate identify the species of origin of Hierodula sp., which is currently adulterated with H. patellifera.
5. Conclusions We fully described the morphology of the four ootheca comprising Mantidis Oötheca. The morphological and statistical analysis revealed that the emergence area, outline, angle of distal end, width of air-filled layer, and weight are useful diagnostic characteristics. Moreover, most morphological characteristics are consistent with the species level and correspond with CO1 barcoding data. The diversity of morphological characteristics may be considered as an identification marker for distinguishing authentic Mantidis Oötheca. Our morphological and molecular data should be help in the accurate identification and quality control of Mantidis Oötheca. In addition, these data will be valuable for future research to understand the
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evolution of these characteristics in a phylogenetic context and the ecology of the praying mantises.
Conflicts of interest The authors declare that they have no conflict of interest or competing financial interests related to the publication of this paper.
Acknowledgment This work was supported by the Establishment of Application Base for Chung-bu Medicinal Materials Described in the Dong Ui Bo Gam from the Korea Institute of Oriental Medicine [grant number KSN1812410].
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Hirase, S., Takeshima, H., Nishida, M., Iwasaki, W., 2016. Parallel mitogenome sequencing alleviates random rooting effect in phylogeography. Genome Biol. Evol. 8 (4), 1267– 1278. https://doi.org/10.1093/gbe/evw063. Jia, K., Jia, T., 2016. Comparison of antidiuretic activity of Ootheca Mantidis before and after processing and its medicinal part against insufficiency of kidney-yang and diuresis rats. China Pharm. 27 (7), 879–881. Kim, H.Y., Lee, Y.J., Ahn, Y.M., Tan, R., Park, J.H., Choi, E.S., Lee, S.H., Lee, H.S., Kang, D.K., Lee, H.S., 2016. Effect of mantidis ootheca on the mechanism of vasorelaxation in thoracic aorta. FASEB J. 30 (1), 945.9. Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16 (2), 111–120. https://doi.org/10.1007/BF01731581. Korea Institute of Oriental Medicine, 2019. Defining Dictionary for Medicinal Herbs. Available from: http://boncho.kiom.re.kr/codex (accessed 4 April 2019). Kramer, K.J., 1973. Oothecal proteins of the oriental praying mantid Tenodera sinensis. Insect Biochem. 3 (11), 297–302. https://doi.org/10.1016/0020-1790(73)90060-7. McCune, B., Mefford, M.J., 2011. PC-ORD. Multivariate Analysis of Ecological Data. Version 6. MjM. Software, Gleneden Beach, Oregon, U.S.A. Ming-zhe, X.U., 2012. Anti-atherosclerotic activities of two compounds from Mantidis Ootheca. J. Anhui Agri. Sci. 32, 57. National Institute of Food and Drug Safety Evaluation, 2012. The Dispensatory on the Visual and Organoleptic Examination of Herbal Medicine. National Institute of Food and Drug Safety Evaluation. Cheongwon, Korea, pp. 341–342.
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Mantidis Oötheca species identificaiton
Patel, S., 2018. Biological of Statilia maculata (Thunberg) (Insecta: Mantodea: Mantidae). J. Adv. Zool. 39 (1), 1–5. Rivera, J., Svenson, G.J., 2016. The Neotropical ‘polymorphic earless praying mantises’–Part I: molecular phylogeny and revised higher‐level systematics (Insecta: Mantodea, Acanthopoidea). Syst. Entomol. 41 (3), 607–649. https://doi.org/10.1111/syen.12178. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30 (12), 2725–2729. https://doi.org/10.1093/molbev/mst197. Tan, Z., Lei, Y., Zhang, B., Huang, L., 1997. Comparison of pharmacological studies on Ootheca Mantidis. China Journal of Chinese Materia Medica 22 (8), 496–499. The Korea Food and Drug Administration, 2014. The Korean Pharmacophoeia, eleventh ed, Volume 2. Korea Food and Drug Administration. Seoul, Korea, pp. 31–32. Wang, X., Hou, F.X., Wang, Y.X., Li, J.D., Yuan, Y., Peng, C., Guo, J.L., 2015. Identification of original species of Mantidis Oötheca (Sangpiaoxiao) based on DNA barcoding. China Journal of Chinese Materia Medica 40 (20), 3963–3966. Wen, L.L., Wan, D.G., Ren, Y., Li, J.D., Guo, J.L., 2013. Corresponding relationship between Mantis and Mantidis oötheca (Sangpiaoxiao). China Journal of Chinese Materia Medica 38 (7), 966–968. Ye, F., Lan, X.E., Zhu, W.B., You, P., 2016. Mitochondrial genomes of praying mantises (Dictyoptera, Mantodea): rearrangement, duplication, and reassignment of tRNA genes. Sci Rep. 6, 25634. https://doi.org/10.1038/srep25634. Zhang, H.L., Ye, F., 2017. Comparative mitogenomic analyses of praying mantises (Dictyoptera, Mantodea): origin and evolution of unusual intergenic gaps. Int. J. Biol. Sci. 13 (3), 367–382, https://doi.org/10.7150/ijbs.17035. 21
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Mantidis Oötheca species identificaiton
Figure captions
Fig. 1. Morphological characteristics of the ootheca used for measurements. (A) Tenodera angustipennis. (B) Hierodula patellifera. (C) Tenodera sinensis. AC: width of air-filled layer. DA: angle of distal end. LE: length of emergence area. LG, length. NE: number of egg chambers. TH, thickness. WD: width. WE: width of emergence area. Asterisks (*) indicate points of attachment to substrate. Scale bars = 1 cm.
Fig. 2. Stereomicroscope micrographs showing the ootheca morphology of four species. (A– C) Tenodera angustipennis. (D–F) Tenodera sinensis. (G–I) Hierodula patellifera. (J–L) Hierodula sp. A, D, G, J. Dorsal view. B, E, H, K. Lateral view. C, F, I, L. Cross-sectional shape. Scale bars = 1 cm.
Fig. 3. Stereomicroscope micrographs showing the emergence area and surface of ootheca. (A–B) Tenodera angustipennis. (C–D) Tenodera sinensis. (E–F) Hierodula patellifera. (G– H) Hierodula sp. A, C, E, G. The shape of emergence area. B, D, F, H. Surface pattern on lateral view. Scale bars = 1 mm.
Fig. 4. Principal component analysis (PCA) performed using the eleven quantitative ootheca morphological variables from four species. AC: width of air-filled layer. DA: angle of distal end. LE: length of emergence area. LE/LG. LG: length. NE: number of egg chambers. TH: thickness. WD: width. WE: width of emergence area. WE/WD. WG: weight. open triangle: Tenodera angustipennis (1) (2-19-0001). filled triangle: Tenodera angustipennis (2) (2-19-
23
Mantidis Oötheca species identificaiton
0002). filled circle: Tenodera sinensis (2-09-0025). filled square: Hierodula patellifera (2-180193A). open square: Hierodula sp. (2-18-0193B).
Fig. 5. Comparison of CO1 sequences of Tenodera angustipennis, Tenodera sinensis, Hierodula patellifera, and Hierodula sp.
Fig. 6. Phylogenetic tree of 63 samples of four species and related species using the neighbor-joining statistics with 1,000 bootstrap replicates based on CO1 sequences. ChP, Pharmacopoeia of the People’s Republic of China 2015 edition. KHP, The Korean Pharmacopoeia 11th edition, 2014.
24
Appendix. Oothecae medicinal materials information of accession number for each sample in this molecular study. Asterisked accession number are sequences obtained in this study. Speices
Sequence_ID
site
Voucher No
Accession No.
Tenodera angustipennis (1)
TA(1)-1
China
2-19-0001-1
MK829263*
Tenodera angustipennis (1)
TA(1)-2
China
2-19-0001-2
MK829264*
Tenodera angustipennis (1)
TA(1)-3
China
2-19-0001-3
MK829265*
Tenodera angustipennis (1)
TA(1)-4
China
2-19-0001-4
MK829266*
Tenodera angustipennis (1)
TA(1)-5
China
2-19-0001-5
MK829267*
Tenodera angustipennis (1)
TA(1)-6
China
2-19-0001-6
MK829268*
Tenodera angustipennis (1)
TA(1)-7
China
2-19-0001-7
MK829269*
Tenodera angustipennis (1)
TA(1)-8
China
2-19-0001-8
MK829270*
Tenodera angustipennis (1)
TA(1)-9
China
2-19-0001-9
MK829271*
Tenodera angustipennis (1)
TA(1)-10
China
2-19-0001-10
MK829272*
Tenodera angustipennis (1)
TA(1)-11
China
2-19-0001-11
MK829273*
Tenodera angustipennis (1)
TA(1)-12
China
2-19-0001-12
MK829274*
Tenodera angustipennis (1)
TA(1)-13
China
2-19-0001-13
MK829275*
Tenodera angustipennis (1)
TA(1)-14
China
2-19-0001-14
MK829276*
Tenodera angustipennis (2)
TA(2)-1
China
2-19-0002-1
MK829277*
Tenodera angustipennis (2)
TA(2)-2
China
2-19-0002-2
MK829278*
Tenodera angustipennis (2)
TA(2)-3
China
2-19-0002-3
MK829279*
Tenodera angustipennis (2)
TA(2)-4
China
2-19-0002-4
MK829280*
Tenodera angustipennis (2)
TA(2)-5
China
2-19-0002-5
MK829281*
Tenodera angustipennis (2)
TA(2)-6
China
2-19-0002-6
MK829282*
Tenodera angustipennis (2)
TA(2)-7
China
2-19-0002-7
MK829283*
Tenodera angustipennis (2)
TA(2)-8
China
2-19-0002-8
MK829284*
Tenodera angustipennis (2)
TA(2)-9
China
2-19-0002-9
MK829285*
Tenodera angustipennis (2)
TA(2)-10
China
2-19-0002-10
MK829286*
Tenodera angustipennis (2)
TA(2)-11
China
2-19-0002-11
MK829287*
Tenodera angustipennis (2)
TA(2)-12
China
2-19-0002-12
MK829288*
Tenodera angustipennis (2)
TA(2)-13
China
2-19-0002-13
MK829289*
Tenodera angustipennis (2)
TA(2)-14
China
2-19-0002-14
MK829290*
Tenodera angustipennis (2)
TA(2)-15
China
2-19-0002-15
MK829291*
Tenodera angustipennis
C3
China
-
KR052038
Tenodera sinensis Tenodera sinensis
TS-1
China
2-09-0025-1
MK829292*
TS-2
China
2-09-0025-2
MK829293*
Tenodera sinensis
TS-3
China
2-09-0025-3
MK829294*
Tenodera sinensis
TS-4
China
2-09-0025-4
MK829295*
Tenodera sinensis
TS-5
China
2-09-0025-5
MK829296*
Tenodera sinensis
TS-6
China
2-09-0025-6
MK829297*
Tenodera sinensis
TS-7
China
2-09-0025-7
MK829298*
Tenodera sinensis
TS-8
China
2-09-0025-8
MK829299*
Tenodera sinensis
-
China
-
KY689132
Hierodula formosana
-
Taiwan
Jftl-1
KR703238
Hierodula patellifera
HP-1
China
2-19-0003A-1
MK829300*
Hierodula patellifera
HP-2
China
2-19-0003A-2
MK829301*
a
b c
Hierodula patellifera
HP-3
China
2-19-0003A-3
MK829302*
Hierodula patellifera
HP-4
China
2-19-0003A-4
MK829303*
Hierodula patellifera
HP-5
China
2-19-0003A-5
MK829304*
Hierodula patellifera
HP-6
China
2-19-0003A-6
MK829305*
Hierodula patellifera
HP-7
China
2-19-0003A-7
MK829306*
Hierodula patellifera
HP-8
China
2-19-0003A-8
MK829307*
Hierodula patellifera
HP-9
China
2-19-0003A-9
MK829308*
Hierodula patellifera
HP-10
China
2-19-0003A-10
MK829309*
Hierodula patellifera
HP-11
China
2-19-0003A-11
MK829310*
Hierodula patellifera
-
China
-
KX611803
Hierodula sp.
HS-1
China
2-19-0003B-1
MK829311*
Hierodula sp.
HS-2
China
2-19-0003B-2
MK829312*
Hierodula sp.
HS-3
China
2-19-0003B-3
MK829313*
Hierodula sp.
HS-4
China
2-19-0003B-4
MK829314*
Hierodula sp.
HS-5
China
2-19-0003B-5
MK829315*
Hierodula sp.
HS-6
China
2-19-0003B-6
MK829316*
Hierodula sp.
HS-7
China
2-19-0003B-7
MK829317*
Hierodula sp.
HS-8
China
2-19-0003B-8
MK829318*
Hierodula sp.
HS-9
China
2-19-0003B-9
MK829319*
Hierodula sp.
HS-10
China
2-19-0003B-10
MK829320*
Hierodula sp.
HS-11
China
2-19-0003B-11
MK829321*
Hierodula sp.
HS-12
China
2-19-0003B-12
MK829322*
Hierodula sp.
H4
China
-
KR011164
Mantis religiosa
-
-
-
FJ802846
d
a
Statilia apicalis
-
-
-
KR011166
Statilia maculata
-
-
-
KT036560
-
-
NC014887
† a
-
Acrida cinerea b
c
d
†, outgroup. Wang et al. (2015). Zhang et al. (2018). Tian et al. (2015). Zhang and Ye (2017).
Table 1. Overview of representative morphological characteristics of oothecae in four Mantis species.
Characters
Tenodera angustipennis (1)
Tenodera angustipennis (2)
Tenodera sinensis
Hierodula patellifera
Hierodula sp.
Shape, outline
fu
fu
ba
el
el
obo
obo
cir to orb
cir
cir
D br/du
D br/du
L br to Y br/sh
R D br to R br/sh
R D br to D br/sh
Length (LG, mm)
34.37 ± 2.96
33.3 ± 4.19
27.9 ± 3.98
26.87 ± 2.93
25.44 ± 2.53
Width (WD, mm)
9.80 ± 0.50
9.87 ± 0.93
23.7 ± 1.97
12.05 ± 1.01
13.3 ± 0.59
Thickness (TH, mm)
9.15 ± 0.91
9.03 ± 0.98
19.1 ± 3.42
10.96 ± 0.84
14.1 ± 0.61
Length of emergence area (LE, mm)
25.14 ± 2.42
25.13 ± 3.44
22.0 ± 3.41
19.04 ± 2.95
22.7 ± 2.46
Width of emergence area (WE, mm)
3.92 ± 0.56
4.11 ± 0.62
6.11 ± 1.01
4.24 ± 0.51
4.23 ± 0.32
fl
fl
fl
fl
so
Number of egg chambers (NE, no)
24.6 ± 2.68
24.1 ± 3.66
20.9 ± 2.98
17.9 ± 1.92
16.9 ± 2.16
Angle of distal end (DA, ˚)
38.8 ± 5.99
39.9 ± 6.82
85.9 ± 8.57
61.3 ± 5.09
91.7 ± 5.14
Width of air-filled layer (AC, mm)
0.69 ± 0.19
0.63 ± 0.10
6.62 ± 1.32
1.09 ± 0.31
0.62 ± 0.16
0.452 ± 0.081
0.418 ± 0.124
0.827 ± 0.17
0.58 ± 0.11
1.04 ± 0.17
Cross-sectional shape Color/shiny
Opening of egg chamber
Weight (WG, g)
Note: All quantitative characters show (abbreviation, unit). Numbers refer to means ± S.D. (sample size n = 100). Shape: ba, barrel-like; el, ellipsoid; fu, fusiform. Cross-sectional shape: cir, circular; obo, obovate; orb, orbicular. Color/shiny: br, brown; D, dark; du, lusterless; L, light; R, reddish; sh, lustrous; Y, yellowish. Opening of egg chamber: fl, flexible flap; so, single opening.
Table 2. The results of the principal component analysis of the eleven quantitative oothecae morphological characters of four species from medicinal materials. PC 1
PC 2
PC 3
(49.884%)
(20.271%)
(14.288%)
No.
Characteristics
1
LG
0.6554
-0.6541
-0.0187
2
WD
-0.8794
-0.3881
-0.2071
3
TH
-0.927
-0.2365
-0.0357
4
LE
0.3627
-0.6812
0.6064
5
WE
-0.5969
-0.5881
-0.1919
6
LE/LG
-0.3998
-0.0018
0.8353
7
WE/WD
0.7811
-0.0827
0.0485
8
NE
0.5377
-0.7174
0.0434
9
DA
-0.9179
0.1571
0.2667
10
AC
-0.7193
-0.4891
-0.4041
11
WG
-0.7198
-0.0085
0.4315
5.487
2.230
1.572
Eigenvalue
Note: The first three PCs with eigenvalue larger than one were represented. The percentages in parentheses indicate the amount of variation explained by each PC, and the components that were loaded most highly for each character are in bold. Total explanation power 84.443 %.
Table 3. Statistical characteristics of CO1 regions among studied species. Species Tenodera angustipennis Tenodera sinensis Hierodula patellifera Hierodula sp.
Amplicon Length (bp) 694 694 694 694
Intra-Species Distance (%) 0.16 ± 0.15 0.41 ± 0.22 0.20 ± 0.14 0.10 ± 0.09
G+C (%) 33.32 31.55 32.73 32.78
Table 4. Nucleotide sequence distance matrix generated by the Kimura-2-parameter (K2P) distance model among all studied species except outgroup, based on the CO1 mitochondrial gene. S. apicalis
S. maculata
-
-
-
-
-
-
-
-
-
-
-
-
13.67
-
-
-
-
-
14.47
13.37
1.93
-
-
-
-
16.25
14.04
17.12
15.46
15.56
-
-
-
S. apicalis
17.91
14.84
16.89
16.75
16.61
13.78
-
-
S. maculata
18.12
15.05
16.68
16.96
16.82
13.98
0.50
-
(%)
Hierodula sp. H. formosana M. religiosa
T. angustipennis
T. sinensis
H. patellifera
-
-
-
-
-
7.22
-
-
-
H. patellifera
14.90
13.43
-
Hierodula sp.
17.67
14.98
H. formosana
15.86
M. religiosa
T. angustipennis T. sinensis